Academic and commercial interest in nucleic acid diagnostics has, to date, focused on qualitative assays. This type of assay determines the presence or absence in a patient sample of a specific gene mutation or infectious pathogen. Molecular assays which achieve these goals are well known. Many rely on amplification techniques, known to those skilled in the art such as the polymerase chain reaction (PCR), NASBA or 3SR, with or without hybridization probing. Others such as Digene Hybrid Capture Assays (DiGene Diagnostics Inc.) do not require amplification prior to detection and are generally less sensitive. Assays have been developed for many infectious pathogens such as Chlamydia trachomatis, Human Immunodeficiency Virus Type 1 (HIV-1) and Type 2 (HIV-2), and human papilloma virus (HPV). Some of these tests have been launched commercially by Roche Diagnostic Systems, Abbott Laboratories and others.
Quantitative assays of nucleic acid analytes also prove useful in diagnosis of a variety of medical disorders. For example, viral load in HIV infection may be correlated with increased risk of clinical progression of HIV disease (Mellors. J. W. et al. (1995). Quantitation of HIV-1 RNA in plasma predicts outcome after seroconversion. Ann. Intern. Med. 122: 573-579). While this example is best known, other quantitative applications also have clinical and commercial interest, such as quantitation of human papilloma virus in PAP smears. (Cuzick, J. et al. (1994) Type-specific human papillomavirus DNA in abnormal smears as a predictor of high-grade cervical intraepithelial neoplasia. Br. J. Cancer 69:167-171; Bavin P. J. et al. (1993) Use of semi-quantitative PCR for human papillomavirus DNA type 16 to identify women with high grade cervical disease in a population presenting with a mildly dyskaryotic smear report. Br. J. Cancer 67:602-605.)).
Notwithstanding their usefulness, quantitative assays of nucleic acid analytes have lagged behind in development. The delay may in part be attributed to technology barriers. Most instruments and methods provide inadequate dynamic range for measuring quantities, thus requiring labor intensive techniques such as multiple serial dilutions and repeat reactions. Further, until recently, PCR methods have been perceived as unreliable for quantitation due to the possibility of contamination and non-linear enzyme kinetics.
The AMPLICOR HIV-1 MONITOR® (Roche Molecular Systems) test is a quantitative molecular assay for HIV RNA levels in blood. The assay is performed on HIV-1 and a subset of HIV-2 RNA found in 200 uL of blood plasma. The RNA is purified from the plasma sample, reverse transcribed and amplified by PCR. The reaction products are quantified by a probe based photometric assay and compared to the levels of a control RNA of known quantity that is added to the plasma sample. The control RNA is reverse transcribed along with the sample RNA and co-amplified using the same amplification primers. Six serial dilutions are necessary to detect across the full range of detectable viral load: 400 copies to 750,000 copies per ml. The test requires that for samples over 750,000 copies, (over 2.2 million copies per ml have been detected) the original patient sample must be diluted. The AMPLICOR assay therefore quantifies across the full range of possible values by a series of multiple dilutions. The AMPLICOR assay does not determine which sub-type or sub-types of HIV-1 are present, and it does not establish if HIV-2 was amplified.
Other quantitative HIV assays have been reported. Some of these papers include:    Mulder, J et al. Rapid and simple PCR assay for quantitation of human immunodeficiency virus type 1 RNA in plasma: Application to acute retroviral infection. J. Clin. Micro. 32:292-300    Dewar, R. L. et al, 1994 Application of branched DNA signal amplification to monitor human immunodeficiency virus type 1 burden in human plasma. J. Infect. Dis. 170:1172-1179    van Gemen, B. et al. 1993 Quantitation of HIV-1-1 RNA in plasma using NASBA during HIV-1-1 primary infection. J. Vir. Meth. 43:177-188.
Each of these prior methods would require the performance of a separate set of reactions for quantitation and sequencing, and even if these methods could be performed concurrently in a single vessel the need for multiple reaction increases the number of reagents required and thus the cost of the procedure.
WO98/41650 discloses a method for quantitative and qualitative analysis of a nucleic acid analyte in a sample suspected to contain the nucleic acid analyte. The method comprises the steps of:
(a) combining the sample with a control nucleic acid, and two primer pairs,                a first primer pair effective to amplify a conserved region of the nucleic acid analyte if present in the sample to produce a conserved fragment having a first length and to amplify the control nucleic acid to produce a control fragment having a second length different from the first length, one member of the first primer pair being labeled with a detectable label, and        a second primer pair effective to amplify a second region of the nucleic acid analyte to produce a sequencing fragment, one member of the second primer pair being labeled with a label effective to permit capture of the primer;        
(b) amplifying the sample and control nucleic acid using the first and second primer pairs to produce an amplification product mixture containing conserved fragments, sequencing fragments and control fragments when the nucleic acid analyte is present in the sample, and only control fragment when the nucleic acid analyte is not present in the sample;
(c) analyzing the relative amounts of conserved fragments and control fragments in the amplification product mixture to quantify the amount of nucleic acid analyte in the sample; and
(d) determining the sequence of the sequencing fragment in the amplification mixture to determine the qualitative characteristics of any nucleic acid analyte in the sample. Thus, although the methodology of WO98/41650 permits sequencing and quantitation in a common reaction, it utilizes different primer pairs for the generation of products evaluated for sequencing and quantitation.